Hops have long been used in brewing to impart bitterness, flavor, foam, and preservatives in beer. The hop plant, humulus lupulus, produces hop cones. These cones contain soft resins, which may be extracted by solvents such as CO2 and ethanol. The extract contains alpha(α)-acids, beta(β)-acids, hop oils, and other unknown substances, which have widely been used in brewing. In the brewing process, the α-acids undergo an isomerization reaction in boiling wort to produce iso-α-acids which contribute bitterness, foam, and antimicrobial activity in finished beer. Most of the β-acids are precipitated and discarded as a by-product due to their low solubility, with no bitterness or flavor significance in beer.
Naturally occurring hop acids may be classified and defined into three classes: a group of six-membered-ring containing α-acids (humulones), five-membered-ring containing isomerized α-acids (iso-α-acids or isohumulones), and six-membered-ring containing β-acids (lupulones) (see FIGS. 1 and 2). Each class also contains at least five analogous forms, differentiated with one side-chain with an R subgroup (isopropyl, isobutyl, sec-butyl, isopentyl, and ethyl; corresponding to a prefix of co, n, ad, pre, and post, respectively). For example, the α-acids consist of cohumulone, (n)-humulone, adhumulone, prehumulone, and posthumulone with corresponding isomerized derivatives as isocohumulone, isohumulone, isoadhumulone, isoprehumulone, and isoposthumulone. In the same manner, the β-acids consist of colupulone, (n)-lupulone, adlupulone, postlupulone, and prelupulone. Typically, hops are extracted with liquid or supercritical CO2 to an extract consisting of α-acids, β-acids, hop oils, and unknown waxes. Cowles et al. (U.S. Pat. No. 4,590,296), hereby incorporated by reference, teaches a method of separating α-acids, β-acids and hop oils from the CO2 hop extract in aqueous solutions by a pH partitioning technique. The α-acids are then converted to iso-α-acids by heat and alkali in an aqueous suspension, typically in the presence of magnesium ions to accelerate the isomerization reaction (Koller et al. U.S. Pat. No. 4,234,516), hereby incorporated by reference, and acidified to afford a pure iso-α-acids resin. Alternatively, Laws et al. (U.S. Pat. No. 4,212,895), Baker et al. (U.S. Pat. No. 4,247,483), Baker (U.S. Pat. No. 4,342,791), Smith & Wilson (U.S. Pat. No. 5,370,897), all of which are hereby incorporated by reference, teach the α-acids present in the CO2 extract may be pre-isomerized to iso-α-acids and then separated from the mixture of β-acids, hop oils and waxes by a pH partitioning technique.
Beers brewed with natural iso-α-acids packaged in clear or green bottles are susceptible to light and UV damages to generate unpleasant skunk or light struck flavors. These flavors have been attributed to the formation of 3-methyl-2-butene-2-thiol (3M2B1T) via a photolysis reaction between iso-α-acids and naturally occurring sulfur compounds in beer (Huvarer, et al. J. Agric. Food Chem. 2005, 53, 1489-1494), hereby incorporated by reference. An isohexenoyl side chain on the iso-α-acids moiety is susceptible to light degradation to generate 3M2B1T and related sulfur compounds. The 3M2B1T has extremely low thresholds at about 2 to 4 ng/L (part per trillion) in lager beers to be perceived as undesired. Furthermore, iso-α-acids are found to be unstable during storage with the same isohexenoyl side chain sensitive to oxidative and non-oxidative degradation to produce stale flavors perceived as papery or cardboard. The oxidation reaction of iso-α-acids in beer also causes a loss of bitterness values and shortens the shelf life of beer. A recent report indicated that trans-iso-α-acids (one isomer of iso-α-acids) have been verified to degrade via a cyclization reaction and leads to a lingering, harsh, and astringent bitter tastes during storage (Intelmann, et al. J. Agric. Food Chem. 2011, 59, 1939-1953), hereby incorporated by reference. In recent years, new technologies have resulted in advanced hop products, which are both light and oxidation resistant hop bittering compounds mostly derived from α-acids and iso-α-acids (Ting et al. J. Am. Soc. Brew. Chem. 54(2):103-109, 19960), hereby incorporated by reference. Goldstein et al. (U.S. Pat. Nos. 4,324,810 & 4,767,640) and Gimble et al. (U.S. Pat. No. 7,087,256), all of which are hereby incorporated by reference, teach methods of making light stable dihydro iso-α-acids (or rho-iso-α-acids) by sodium borohydride (NaBH4) reduction via a one or two-step process. The key steps for making advanced products involve either a reduction of a carbonyl (C═O) to a hydroxyl (CH—OH) on the isohexenoyl side chain to form dihydro iso-α-acids (rho-iso-α-acids), or a hydrogenation of(C═C) double bonds to (CH—CH) single bonds to form tetrahydro α-acids, tetrahydro iso-α-acids, or combining both treatments to form hexahydro iso-α-acids (see FIG. 1). These treatments affect the properties of iso-α-acids toward light and oxidative stabilities, sensorial bitterness, foam retention, as well as anti-microorganism strength. The light stable iso-α-acids derivatives or advanced hop bittering products have been commonly used for brewing light stable beer, enhancing foam, adjusting bitterness, and strengthening anti-microorganisms. Their effects are in a relative order of tetrahydro>hexahydro>iso-α-acids. Of the advanced products, tetrahydro iso-α-acids and hexahydro iso-α-acids are more valuable and important than the others in brewing. They are also applicable for non-brewing usages in other beverages, health supplements and foods. For example, Barney et al. (U.S. Pat. No. 5,455,038), hereby incorporated by reference, discovered that tetrahydro isohumulone and hexahydro colupulone are superior agents for inhibiting Listeria than beta-acids. Tripp et al. (U.S. Pat. No. 7,901,713), hereby incorporated by reference, discloses a method of treating an inflammatory condition comprising a therapeutically effective amount of dihydro iso-α-acids, tetrahydro iso-α-acids, and hexahydro iso-α-acids.
The original resins or free acid forms of α-acids, iso-α-acids, dihydro iso-α-acids, tetrahydrop iso-α-acids, and hexahydro iso-α-acids are viscous liquids, except β-acids which are solids having a melting point between 85° to 93° C. They are less soluble in water but soluble in most organic solvents as well as liquid/supercritical CO2. They are also soluble in alkaline aqueous solutions as salt forms and are commercially available in 5%-40% (w/w) aqueous solution. The salt forms are possibly dissociated in alcohols.
A typical hydrogenation reaction simply adds hydrogen to a double or triple bond connecting two atoms in the structure. Thus, hop acids containing multiple unsaturated (C═C) side chains are applicable to be hydrogenated with hydrogen and a hydrogenation catalyst to the hydrogenated hop acids. The hop acids are commonly performed in conventional organic, alcoholic, CO2 and/or aqueous solvent in one form or another. One strategy of making tetrahydro iso-α-acids and hexahydro iso-α-acids starts from the α-acids and the chemistry is illustrated in FIG. 1.
Several methods of preparing tetrahydro iso-α-acids from the α-acids, rather than from the less valuable β-acids, are disclosed. Anteunis & Verzele (Bull. Soc. Chim. Belg. Vol. 68, 1959, 315-324), hereby incorporated by reference, teaches a hydrogenation of α-acid (humulone) in methanol to tetrahydro iso-α-acid (tetrahydrohumulone). Brown, Howard & Tatchell (J. Chem. Soc. 1959, 545-551), hereby incorporated by reference, teaches an isomerization of hydrogenated α-acids to corresponding tetrahydro iso-α-acids in ethanolic alkali solution and a hydrogenation of isomerized α-acids in ethanol. However, the hydrogenation of the α-acids reaction in methanol or ethanol often causes undesirable hydrogenolyzed (cleaved) α-acids by-products. Hay & Homiski (J. Agric. Food Chem. 1991, 39, 1732-1734), Hay (U.S. Pat. No. 5,013,571), and Poyner et al. (U.S. Pat. No. 5,600,012), all of which are hereby incorporated by reference, disclose the hydrogenation of iso-α-acids in aqueous, aqueous alcohol or chlorinated hydrocarbon solution to form tetrahydro iso-α-acids. Stegink et al. (U.S. Pat. No. 5,296,637), hereby incorporated by reference, demonstrates a hydrogenation of α-acids as alkaline metal salts in aqueous or alcoholic solution followed by isomerization. Ting et al. (U.S. Pat. Nos. 5,523,489; 5,767,319; 5,874,633 and 6,303,824), all of which are hereby incorporated by reference, claim the hydrogenation of iso-α-acids or their metal salts in either aqueous alcohol or alcohol solutions to form tetrahydro iso-α-acids. Ting et al. (U.S. Pat. No. 6,020,019) and Wilson & Smith (U.S. Pat. No. 7,344,746, EP 1230337, WO 2001036581, & CA 2391973), all of which are hereby incorporated by reference, disclose the processes of hydrogenation of hop acids in liquid/supercritical CO2 to produce high purity of tetrahydro iso-α-acids and hexahydro iso-α-acids. The prior art teach a process of making tetrahydro iso-α-acids via either a hydrogenation of α-acids to tetrahydro α-acids followed by isomerization or a reversed process by isomerization of α-acids to iso-α-acids followed by the hydrogenation. The disadvantage is using and recycling the solvents.
The preparation of hexahydro iso-α-acids are an easier task involving either a chemical reduction of the carbonyl (C═O) to a hydroxyl (CH—OH) on the isohexenoyl side chain of iso-α-acids using sodium borohydride before the hydrogenation, or reversal of the process. Worden & Todd (U.S. Pat. No. 3,552,975), hereby incorporated by reference, discloses a method of preparing hexahydro iso-α-acids using alkali metal borohydride reduction of tetrahydro iso-α-acids in organic solvents. Hay (U.S. Pat. No. 5,013,571), hereby incorporated by reference, teaches a reversal of the necessary reducing agents from α-acids or iso-α-acids to hexahydro iso-α-acids. Wilson & Smith (U.S. Pat. No. 7,344,746, EP 1230337 and CA 2391973), all of which are hereby incorporated by reference, claim a method of hydrogenation of dihydro iso-α-acids to hexahydro iso-α-acids in a solvent-free or CO2 system. Mertens et al. (EP 2580312), hereby incorporated by reference, discloses a method of employing ruthenium-containing catalyst that catalyzes the hydrogenation of both C═C and C═O bonds on the side-chain of iso-α-acids or tetrahydro iso-α-acids to produce hexahydro iso-α-acids either in a solvent-free condition, or in the presence of a solvent phase (e.g., carbon dioxide, water, ethanol, organic solvent, or mixtures thereof).
Another strategy of making tetrahydro iso-α-acids and hexahydro iso-α-acids may be prepared from β-acids (lupulones) and the chemistry is illustrated in FIG. 2. It is valuable because the β-acids are normally discarded as by-products in hops and brewing. Both α-acids and β-acids have a common six-membered ring base structure that differs in that where the α-acids have an active hydroxyl (OH) group while the β-acids have an inactive five-carbon alkenyl group at C-4 position. The mechanisms of preparing tetrahydro iso-α-acids from the β-acids are different from that of the α-acids, in which one of the vicinal isopentyl (5 carbon alkenyl) side-chains on the β-acids moiety is cleaved to form desoxytetrahydro α-acids (retaining a six-membered ring structure) by a hydrogenolysis reaction and then substituted with an OH group by an oxidation to form tetrahydro α-acids prior to the isomerization reaction. The hydrogenolysis adds hydrogen and results in dissociation (breaking up) of the molecule (or called destructive hydrogenation). The hydrogenation and hydrogenolysis reaction is usually performed under mild and controllable conditions because the reaction is exothermic and fast. It is commonly practiced with hydrogen and a noble metal hydrogenation catalyst in the presence of solvents. Alcoholic and organic solvents have been used for the hydrogenation and hydrogenolysis for many years as solvents. Most commonly used active oxidation agents are oxygen, air, hydrogen peroxide, and peracids, in which the oxidation occurs in air as an auto-oxidation.
Worden & Todd (U.S. Pat. No. 3,552,975), hereby incorporated by reference, teaches a method of hydrogenolysis of β-acids using palladium on carbon and hydrogen in an acidic methanol (adding hydrochloric acid or HCl) to form desoxytetrahydro α-acids (intermediates or precursors of tetrahydro α-acids); then oxidizing the intermediates with air in the presence of lead salt to produce lead salts of tetrahydro α-acids and isomerize the free tetrahydro α-acids in an alkaline solution to the final tetrahydro iso-α-acids. The hydrogenolysis takes place in methanol with adding corrosive HCl. The final product is a crude mixture from which the lead residues can only be removed with great difficulty. The presence of residual lead in products to be consumed is undesirable. Worden (U.S. Pat. No. 3,923,897), hereby incorporated by reference, discloses an improvement on the prior art (U.S. Pat. No. 3,552,975) using organic peracids to replace lead salt and air in a water immiscible organic solvent and adding magnesium or calcium salts for the isomerization. The process does not utilize lead salt but it is conducted in water immiscible organic solvents and it involves cumbersome solvent changes, which increase process cost. The presence of even residual amounts of such solvents and organic peracids in food products, such as beverages, is undesirable. Cowles et al. (U.S. Pat. No. 4,644,084), hereby incorporated by reference, discloses a method of making tetrahydro iso-α-acids involving a hydrogenolysis of β-acids in a sulfuric acid (H2SO4) added ethanol solution to form desoxytetrahydro α-acids followed by a simultaneous oxidation and isomerization reaction in an aqueous ethanol mixture containing alkaline and magnesium salt and purging with air to form the desired tetrahydro iso-α-acids. Although this art eliminates using organic peracids, lead salt and water immiscible organic solvent, it still uses sulfuric acid, ethanol, magnesium salt, and aqueous ethanol.
The prior art discovered that the hydrogenolysis of the β-acids to desxoytetrahydro α-acids are performed in acidic solvents (commonly uses methanol or ethanol with HCl or H2SO4). Worden & Todd (U.S. Pat. No. 3,552,975), hereby incorporated by reference, claims specifically that the hydrogenolysis of the β-acids in a pH not greater than 1 in alcohols or ether, otherwise, favors the hydrogenation of C═C double bonds. Ting et al. (U.S. Pat. No. 6,020,019), hereby incorporated by reference, claims the hydrogenolysis of the β-acids in CO2 occurs with adding an acidic alcohol modifier. Thus under higher pH (no inorganic acids) conditions, the hydrogenation is more dominant and produces dihydro, tetrahydro, or hexahydro β-acids derivatives than the hydrogenolysis to form desoxytetrahydro α-acids. Wilson & Smith (U.S. Pat. No. 7,344,746, EP 1230337, WO 2001036581, & CA 2391973), all of which are hereby incorporated by reference, confirms that a direct hydrogenation of the β-acids or in CO2 resulted in no occurrence of the hydrogenolysis reaction. Furthermore, inorganic acids (HCl, H2SO4 and H3PO4) are very corrosive and laborious removal from the process.
Both hydrogenation and hydrogenolysis are well-known processes, which are commonly employed in many organic chemical synthesis schemes, including the manipulation of the β-acids, the α-acids, and their derivatives. Alcohols and other organic compounds have been used for many years as solvents in these processes. Although ethanol, CO2, or organic solvents may be recovered in practice, the costs of capital equipment and the loss of such solvents (during the process due to handling, reaction, and evaporation), environmental problems and residual solvent contamination of the final product associated with organic solvents are usually unavoidable and significant. One can readily appreciate that a neat (solvent-free) process, which avoids using solvents, inorganic acids, and other artifacts, would be of economical benefits, significantly important, and valuable.
May et al. (U.S. Pat. No. 6,198,004), hereby incorporated by reference, discloses a method of hydrogenation of iso-α-acids in an alkaline aqueous solution to tetrahydro iso-α-acids using a noble metal catalyst wherein, the catalyst is added incrementally or continuously throughout the hydrogenation step. Smith & Wilson (CA 2391973) and Mertens et al. (EP 2580312), both of which are hereby incorporated by reference, disclose a process for direct hydrogenation of the α-acids derivatives in the absence of any conventional organic solvent, CO2, and aqueous to produce tetrahydro iso-α-acids and hexahydro iso-α-acids. Smith & Wilson (CA 2391973), hereby incorporated by reference, discloses a direct hydrogenation of β-acids to produce only the hydrogenated (β-acids derivatives and no hydrogenolyzed products. None of the prior art have disclosed a solvent-free hydrogenolysis of the β-acids to form desoxytetrahydro α-acids. The Smith & Wilson (CA 2391973) and Mertens et al. (EP 2580312) solvent-free methods are limited to the α-acids resin and iso-α-acids derivatives having advantages and benefits of no solvents, lower equipment costs, and higher output than those using conventional solvent processes. It would be highly appreciated if the hydrogenolysis of the β-acids can be performed under a solvent-free and clean system.
A preferred strategy of making tetrahydro iso-α-acids and hexahydro iso-α-acids is from both α-acids and β-acids and the chemistry is illustrated as FIGS. 1, 2 and 3. A Chinese Patent (CN 94100149.0), hereby incorporated by reference, discloses a method of converting both α-acids and β-acids to tetrahydro iso-α-acids involving a hydrogenation and hydrogenolysis of an extract comprising pre-isomerized α-acids (iso-α-acids) and β-acids in ethanol, hydrogen and palladium on carbon as catalyst to simultaneously produce tetrahydro iso-α-acids and desoxytetrahydro α-acids, respectively. The spent palladium catalyst is filtered out. The oxidation and isomerization of desoxytetrahydro α-acids are conducted with magnesium salts and air in an aqueous alkaline ethanol solution similar to the Cowles et al. (U.S. Pat. No. 4,644,084) procedures. It results in total tetrahydro iso-α-acids from both α-acids and β-acids. This method has advantages of utilizing both α-acids and β-acids without adding inorganic acid. However, unavoidably it uses ethanol, aqueous ethanol and magnesium salts. The disadvantages of the prior art (i.e., CN 94100149.0) are complication, costs of recovering ethanol and removing magnesium salt. Particularly, a common problem of using solvents is its residues remaining in the final products, which have concerns of off-flavor or health; for example, a residual esters aroma is often a concern for some brewers due to an esterification of residual methanol or ethanol. It would be desirable to have a method of converting α-acids and β-acids into tetrahydro iso-α-acids without the use of solvents, inorganic acids, or magnesium salt.